Poxviruses, such as vaccinia virus, are unique among DNA viruses in replicating solely within the cytoplasm of the infected cell. As a result, the progression of the viral life cycle is intimately linked to interactions with the organelles, cytoskeleton and cytoplasmic milieu of the host cell. Cytoplasmic replication of poxviruses is also associated with their nearly complete genetic autonomy from the host. Among the ~ 200 gene products encoded by the virus are those mediate gene expression, genome replication and maturation, and the complex process of virion assembly. At its essence, a productive viral infection requires that the viral genome be efficiently replicated and packaged into nascent, infectious virions. It is this process of genome encapsidation, which lies at the interface of replication and morphogenesis, that it is the focus of this exploratory application. Viral DNA synthesis, which is accomplished by a repertoire of virally encoded proteins, is followed by the processing of concatemeric intermediates into mature, monomeric genomes by a virally encoded resolvase. These monomeric genomes are then thought to be condensed and translocated into immature virions (IV) prior to the closure of the IV membrane. Immature virions are delimited by a lipid bilayer and surrounded by an external lattice composed of the viral D13 protein. The interior of the immature virion appears to contain an unstructured pool of proteins that will later form the internal core of the mature virion. Genetic analyses of conditionally lethal viral mutants have identified two viral proteins as being directly associated with genome encapsidation: I6 and A32. In vitro, the I6 protein binds with great specificity to DNA probes representing the telomeric hairpins of the viral genome. These ~50 bp telomeres, which are covalently closed due to the presence of a short hairpin loop, are highly A-T rich (>90%) and contain extrahelical bases (EHBs) on both DNA strands. The binding of I6 to telomeric probes is dependent upon the presence of the EHBs. The A32 protein, in contrast, is thought to belong to the FtsK-HerA family of pumping ATPases, as defined by the conservation of characteristic sequence motifs. We are hypothesizing that the genome is condensed by association with polyamines, that I6 marks mature genomes for encapsidation by binding to their telomeres, and that subsequent interactions between I6 and A32 lead to activation of A32's ATPase activity and translocation of the genome into the immature virion. This hypothesis will be tested in two Aims. Aim I will explore the condensation of the viral genome and its association with I6;Aim II will explore the role of A32 as a translocating ATPase. PUBLIC HEALTH RELEVANCE: The fear that smallpox might be used as a bioterrorist weapon, and the recognition that monkeypox virus is a cause of human morbidity and mortality, has reinforced the need to study the poxviral life cycle in depth. Our investigations of genome encapsidation will provide fundamental insights into this poorly understood facet of the viral life cycle and define new targets for the development of rational antiviral therapies.